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. 2017 Sep 26;37(20):e00226-17.
doi: 10.1128/MCB.00226-17. Print 2017 Oct 15.

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

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Free PMC article

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

Jaewon Min et al. Mol Cell Biol. .
Free PMC article

Abstract

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

Keywords: ALT; G quadruplex; MiDAS; R-loop; RAD51; RAD52; SMC5/6; TIMELESS; cyclin E; telomerase; telomere.

Figures

FIG 1
FIG 1
Mitotic DNA synthesis at telomeres in ALT cells is triggered by DNA replication stress. (A) Overview of H1299ALT cell generation from its parental telomerase-positive cancer cell line, H1299 (13). (B and C) Fragile telomere analysis of H1299 and H1299ALT with or without PDS (pyridostatin) treatment (10 μM). (B) Representative images showing fragile telomeres on metaphase spreads. (C) Quantification of fragile telomeres as a percentage of fragile telomeres (results are from three independent experiments and are given as means ± standard errors of the means [SEM]). (D and E) Metaphase chromosome-telomere dysfunction-induced focus (Meta-TIF) analysis of H1299 and H1299ALT with or without PDS treatment. (D) Representative images showing gamma-H2AX-positive telomeres on metaphase spreads. (E) Quantification of Meta-TIFs as the number of gamma-H2AX-positive telomeres per metaphase spread (results are from two independent experiments and are given as means ± SEM). (F) Experimental scheme for mitotic DNA synthesis analysis (left) and cell cycle analysis for the indicated time points (right). (G and H) Telomeric mitotic DNA synthesis (MiDAS) analysis of H1299 and H1299ALT with or without PDS treatment. (G) Representative images showing mitotic DNA synthesis at telomeres on metaphase spreads. (H) Quantification of telomeric MiDAS and number of EdU-positive telomeres per metaphase spread (results are from three independent experiments and are given as means ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (unpaired Student's t test).
FIG 2
FIG 2
Telomeric MiDAS is a conservative DNA synthesis process mediated by break-induced replication. (A) Proposed model for MiDAS. During MiDAS, break-induced replication leads to conservative DNA synthesis (EdU signal in a single chromatid), whereas homologous recombination-mediated reinitiation leads to semiconservative DNA synthesis (EdU signals in both chromatids). (B and C) Analysis of nontelomeric MiDAS of H1299ALT with PDS treatment. (B) Representative images showing EdU signal in a single chromatid (upper) or EdU signals in both chromatids (bottom). (C) Quantification of nontelomeric MiDAS as a percentage of EdU signal in a single chromatid or in both chromatids (n = 90). (D and E) Analysis of telomeric MiDAS of H1299ALT with PDS treatment. (D) Representative images showing an EdU signal(s) localized at a telomere(s) in a single chromatid (upper) or in both chromatids (bottom). (E) Quantification of telomeric MiDAS as a percentage of the EdU signal(s) localized at a telomere(s) in a single chromatid or in both chromatids (n = 85).
FIG 3
FIG 3
G2/M checkpoint prevents spontaneous telomeric MiDAS in ALT cancer cells. (A) Immunoblot of U2OS, U2OS with SV40 T antigen genomic DNA (LTg), and Saos2 using p53, SV40 large T antigen (T Ag), and actin (control) antibodies. (B and C) Meta-TIF analysis of U2OS, U2OS with LTg, and Saos2. (B) Representative images showing gamma-H2AX-positive telomeres on metaphase spreads. (C) Quantification of Meta-TIFs as the number of gamma-H2AX-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). (D and E) Telomeric MiDAS analysis of U2OS, U2OS with LTg, and Saos2. (D) Representative images showing telomeric MiDAS. (E) Quantification of telomeric MiDAS as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). ***, P < 0.001; ****, P < 0.0001 (unpaired Student's t test).
FIG 4
FIG 4
Replication stresses promote telomeric MiDAS. (A and B) Representative images showing localizations of G-quadruplex (G4, 1H6) (A) and R-loop (DNA-RNA hybrid, S9.6) (B) at telomeres. (C and D) Telomere dysfunction-induced focus (TIF) analysis of Saos2 in the presence of cyclin E overexpression, PDS treatment (10 μM), or siRNaseH1 transfection. (C) Representative images showing gamma-H2AX-positive telomeres. (D) Quantification of TIFs as a percentage of cells containing gamma-H2AX foci at telomeres (two independent experiments with results shown as means ± SEM). (E) Fragile telomere analysis of Saos2 in the presence of cyclin E overexpression, PDS treatment, or siRNaseH1 transfection. Shown is the quantification of fragile telomeres as a percentage of fragile telomeres (two independent experiments with results shown as means ± SEM). (F and G) Telomere clustering analysis of Saos2 in the presence of cyclin E overexpression, PDS treatment, or siRNaseH1 transfection. (F) Representative images showing telomere clustering. (G) Quantification of telomere clustering as the percentage of cells containing clustered telomeres (≥2 μM) (two independent experiments with results shown as means ± SEM). (H and I) APB (ALT-associated PML body) analysis of Saos2 in the presence of cyclin E overexpression, PDS treatment, or siRNaseH1 transfection. (H) Representative images showing the colocalizations of PML and telomeres. (I) Quantification of APBs as a percentage of cells containing PML bodies at telomeres (two independent experiments with results shown as means ± SEM). (J) C-circle assay for Saos2 in the presence of cyclin E overexpression, PDS treatment, or siRNaseH1 transfection. (Left) Quantification of C-circle assay as the relative amount of C-circle assay products. (Right) Slot-blot images for C-circle assay (three independent experiments with results shown as means ± SEM). (K) Telomeric MiDAS analysis of Saos2 in the presence of cyclin E overexpression, PDS treatment, or siRNaseH1 transfection. Shown is the quantification of telomeric MiDAS as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; n.s., nonsignificant (unpaired Student's t test).
FIG 5
FIG 5
Telomere clustering and telomeric MiDAS is suppressed by TIMELESS/TIPIN but is facilitated by SMC5/6. (A) Immunoblot of Saos2 after siRNA transfection targeting TIMELESS or TIPIN. (B and C) Telomere clustering analysis of Saos2 after siRNA transfection targeting TIMELESS or TIPIN. (B) Representative images showing telomere clustering. (C) Quantification of telomere clustering as a percentage of cells containing clustered telomeres (≥2 μM) (two independent experiments with results shown as means ± SEM). (D and E) Telomeric MiDAS analysis of Saos2 after siRNA transfection targeting TIMELESS or TIPIN. (D) Representative images showing telomeric MiDAS. (E) Quantification of telomeric MiDAS as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). (F) Immunoblot of Saos2 after siRNA transfection targeting SMC5/6. (G and H) Telomere clustering analysis of Saos2 after siRNA transfection targeting SMC5/6. (G) Representative images showing telomere clustering. (H) Quantification of telomere clustering as a percentage of cells containing clustered telomeres (≥2 μM) (two independent experiments with results shown as means ± SEM). (I and J) Telomeric MiDAS analysis of Saos2 after siRNA transfection targeting SMC5/6. (I) Representative images showing telomeric MiDAS. (J) Quantification of telomeric MiDAS as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student's t test).
FIG 6
FIG 6
Identification of the molecular mechanism underlying telomeric MiDAS. (A) Representative images showing the telomere localization of PML, phosphorylated S33 RPA, MRE11, RAD52, and RAD51 in interphase cells and metaphase chromosomes of Saos2. (B) Telomeric MiDAS analysis of TIPIN siRNA-transfected Saos2 cells with treatment of inhibitors targeting RAD52 (20 μM), RAD51 (20 μM), ATM (25 μM), ATR (25 μM), CHK1 (25 μM), MRE11 (25 μM), and DNA polymerase (APH; 10 μM). Quantification of telomeric MiDAS is shown as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). (C) Immunoblot of Saos2 after siRNA transfection targeting RAD52, RAD51, or BRCA2. (D) Fragile telomere analysis of Saos2 after siRNA transfection targeting RAD52, RAD51, or BRCA2. Quantification of fragile telomeres is shown as a percentage of fragile telomeres (two independent experiments with results shown as means ± SEM). (E) Meta-TIF analysis of Saos2 after siRNA transfection targeting RAD52, RAD51, or BRCA2. Quantification of Meta-TIFs is shown as the number of gamma-H2AX positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). (F) Telomeric MiDAS analysis of Saos2 after siRNA transfection targeting RAD52, RAD51, or BRCA2. Quantification of telomeric MiDAS is shown as the number of EdU-positive telomeres per metaphase spread (two independent experiments with results shown as means ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant (unpaired Student's t test).
FIG 7
FIG 7
Schematic models for telomeric MiDAS processes.

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